The present invention relates to gas chromatography (GC). More particularly, the present invention relates to method translation, method development automation, method optimization, solute identification, improving reproducibility of elution pattern (elution pattern locking), method porting, and the like.
Gas Chromatography
A typical gas chromatography (GC) system is comprised of a gas chromatograph and a computer controller as described in U.S. Pat. No. 5,405,432. The prime task of a chromatographic analysis of a sample mixture of chemical compounds (also known as analytes or solutes) by a GC system is to separate the solutes from each other, to identify them, and to quantify their amounts.
The separation of individual solutes in a sample mixture takes place in a chromatographic column also described in U.S. Pat. No. 5,405,432. Due to the different interaction of different solutes with the stationary phase in the column, it takes a different amount of time for the different solutes to travel through the column. As a result, the solutes, simultaneously injected in the column as a single mixture, elute from the column at different times, thus causing the separation of the solutes from each other. A detector converts the sequence of the solutes eluting from the column into a chromatogramxe2x80x94a sequence of chromatographic peaksxe2x80x94that can be electronically stored in a computer memory and/or displayed (e.g., electronically, on paper, etc.). Several examples of chromatograms are shown in U.S. Pat. No. 5,405,432.
The identity of each analyte, separated by the column, is typically associated with retention time (also known as elution time), tR, of the corresponding peak in a chromatogram. A typical computer aided identification of the separated solutes makes use of a calibration tablexe2x80x94an electronic list of records for all solutes of interest. Each record contains a solute identification and a retention time of the corresponding peak for that solute. A calibration table may also include information regarding tolerance windows for the retention times, as well as other information. A solute of interest can be correctly identified if its actual retention time in a particular analysis falls within the tolerance window for its retention time. To prevent a misidentification of a given peak with its immediately preceding or a following neighbor, the tolerance windows must have relatively narrow widths. This, in turn, may require a relatively high reproducibility of retention times for all peaks.
As described in U.S. Pat. No. 5,405,432, a solute""s retention time depends on many parameters such as the column length, L, internal diameter, dc, stationary phase film thickness, df, stationary phase type, carrier gas type, column pressure, column flow rate, and column temperature, T. Some of these parameters, such as the gas pressure or flow rate and the column temperature, can either remain constant during a given analysis or can change according to predetermined programs. The column parameters together with the calibration table and with the parameters of other components (injectors, detectors, etc.) of a GC system comprise a particular method of analysis of a particular mixture. Any change in the relevant method parameters can lead to the change in the retention times of some or all peaks. If the retention time changes are not accompanied by the necessary changes in the calibration table, misidentification of some or all solutes can occur.
Generally, it is difficult to predict the retention time changes caused by arbitrary changes in the method parameters. However, there are practically important exceptions. A concept of void time, tM, is useful for the description of these exceptions. The void time is a retention time of a so-called unretained solutexe2x80x94that is, one that does not interact with the stationary phase, and, as a result, travels with the same velocity as the velocity of a carrier gas. Methane is frequently used as an unretained solute in practical measurements of tM.
Method Translation
In some cases, the changes to retention times caused by a change in a method parameter can be predicted. The first case is referred to as method translation and may be applied to methods employing a constant pressure, as well as some other restrictions described below. To illustrate method translation, let T1(t) and T2(t), where t is time since injection of the mixture in a column, be the temperature programs in methods 1 and 2, respectively. Let also tM1,ref and tM2,ref be, respectively, reference void times in methods 1 and 2. These quantities should be isothermally measured at the same reference temperature. If the following conditions are met:
a. the capillary columns have the same type of a liquid stationary phase;
b. the ratio of a column internal diameter and the stationary phase film thickness is the same, i.e. dc1/df1=dc2/df2;
c. column inlet and outlet pressure remain constant during the analysis (this is known as a constant pressure mode); and
d. temperature programs T1(t) and T2(t) relate as T2(t)=T1(Gxc3x97t) where G=tM2,ref/tM1,ref (for a piece-wise linear temperature program, this means that all temperature plateaus in method 2 are G-fold shorter than their counterparts in method 1, and all temperature ramps in method 2 have G-fold higher heating rates than their counterparts in method 1),
then retention time, tR2, of any peak in method 2 can be found as tR2=tR1/G where tR1 is retention time of the peak corresponding to the same solute in method 1. Two methods satisfying this set of conditions are known as mutually translatable methods, and each of the two methods is known as a translation of the other, where quantity G=tM2,ref/tM1,ref is known as a speed gain in method 2 relative to method 1.
While the above-mentioned restrictions disallow some differences in parameters for the methods to be mutually translatable, the restrictions do not prevent many other important differences. Thus, mutually translatable methods may use columns with different diameters and lengths, may use different types of carrier gas (helium, hydrogen, nitrogen, etc.), may have different flow rates as well as different inlet pressures and outlet pressures. The latter includes the cases when, in one of the mutually translatable methods, outlet is at the vacuum while in another the outlet is at an ambient or at any other constant pressure. A theoretical analysis of the method translation is described in Blumberg, L. M. and Klee, M. S., xe2x80x9cMethod Translation and Retention Time Locking in Partition GCxe2x80x9d, Analytical Chemistry, vol. 70, number 18, Sept. 15 1998, pp. 3828-3839. An algorithm for the calculation of the temperature program in a translated method from that of an original method, and from the ratios of the column dimensions and other relevant parameters of the two methods, is also described in U.S. Pat. No. 5,405,432.
Method translation has several useful properties. It can be viewed, for example, as a G-fold compression (stretching, if G less than 1) of the time axis of a temperature program in a translated method compared to that in the original method while keeping the temperature axis unchanged. More specifically, method translation does not change initial and final temperatures preceding and following each temperature ramp. It only reduces the duration of each temperature plateau and increases the heating rate of each temperature ramp by the same factor equal to the speed gain, G.
Method translation has similar time scaling effect on peak retention times. Specifically, it reduces (increases, if G less than 1) all retention times in a translated method by the same speed gain G. This suggests that the calibration table for a translated method can be regenerated from the calibration table of the original method by simply dividing each retention time entry in the original calibration table by the same factor G. The fact that the translated method runs G times faster (if G greater than 1) than the original one, is the reason to refer to G as to the speed gain.
Method translation can be viewed as proportional compression (stretching, if G less than 1) of the chromatographic time axis by a fixed factor equal to speed gain, G, defined as the ratio of original and translated void times measured at the same temperature. Simply stated, method translation allows an increase of the speed of analysis by a factor of G. Practical examples having G higher than 10 are known from the literature.
Another beneficial view of method translation can be described using the concept of a peak retention pattern. Rather than expressing the chromatographic time, t, in absolute time units (seconds, minutes, etc.), one can express it in the units of a reference void time, tM,ref, using dimensionless time x=t/tM,ref instead of t. A temperature program where time is expressed in these dimensionless units is known as a normalized temperature program. As long as the reference void times in all mutually translatable methods are measured at the same temperature, all these methods (a) have the same normalized temperature program and (b) yield the same dimensionless retention time for the same solute.
A sequence of dimensionless retention times for a given sequence of solutes is known as a peak retention pattern for those solutes. As long as the reference void times in the mutually translatable methods are measured at the same temperature, all those methods yield the same retention pattern for the same sequence of solutes. As a result, the dimensionless retention time entries in the calibration tables of the mutually translatable methods are the same, and, hence, there is no need to regenerate those entries for each particular translation of the same method.
Sometimes, rather than expressing retention times, tRA, tRB, tRC, etc. and retention patterns in dimensionless units XRA=tRA/tM,ref, XRB=tRB/tM,ref, XRC=tRC/tM,ref, etc., respectively, it is chromatographically more meaningful and convenient to use dimensionless quantities kRA=tRA/tM,refxe2x88x921, kRB=tRB/tM,refxe2x88x921, kRC=tRC/tM,refxe2x88x921, etc., respectively. These quantities are known as peak retention factors. An additional discussion of peak retention patterns can be found in Blumberg, L. M. and Klee, M. S., xe2x80x9cMethod Translation and Retention Time Locking in Partition GCxe2x80x9d, Analytical Chemistry, vol. 70, number 18, Sept. 15, 1998, pp. 3828-3839. Utilization of retention factor databases for the sample identification in GC is described in U.S. Pat. No 6,153,438.
Dimensionless times can be used for the expression of other time events during a GC analysis. This, in turn, allows for a generic description of GC methods where a description based on the dimensionless time is used as a description of the unique core of all mutually translatable methods. A normalized temperature program and a normalized calibration table would be the components of the core description of a method. The particulars (the column dimensions, the carrier gas type and its inlet and outlet pressure) of the different translatable implementations of the same method can be supplied as conditions in each particular analysis based on the same core method. Manipulation of the particular parameters allows speeding-up or slowing-down the analysis, trading separation for time, or either of those for the sample capacity, etc.xe2x80x94all without affecting the peak retention pattern and a normalized calibration table. This allows, for example, a faster analysis with lower separation for many repetitive trials during the method development, and upgrading of the final analysis to the required separation. This also allows for a transparent pressure optimization (manual and/or automatic) in a temperature programmed analysis as described below.
It is known from GC theory how to approximately find the flow rate (or, equivalently, velocity, pressure, etc.) of a carrier gas that results in the best separation of a given (typically, the most critical) pair of solutes, in a given separation of that pair in the shortest time, etc. This optimization is based on the analysis of the plate height vs. flow rate, plate duration vs. flow rate, and similar curves. An experimental evaluation of these curves allows for an additional fine-tuning of these optimization techniques. Typically, these curves are evaluated only for the isothermal conditions leading to recommendations that might not be optimal under the temperature programmed conditions. A better approach can be to, first, develop a temperature program for some fixed pressure, and then further optimize the pressure for a particular (possibly, most critical) pair of peaks using translatable variations of the pressure. These variations allow for further optimization of the separation without changing the peak retention pattern in the entire chromatogram.
Another benefit of method translation is its use for retention time locking. Several realizations of nominally the same method can yield different retention times for the same solute, and different retention patterns for the same mixture. For example, due to specifics of column manufacturing, two nominally identical columns can have different actual internal diameters and lengths. Used in two nominally identical GC systems these columns can yield different void times even in nominally identical methods. The pressure measurement errors in those GC systems can further magnify the void time difference. That difference in otherwise identical methods can result in a retention pattern difference yielded by these methods. According to the method translation, adjusting the inlet pressure in each nominally identical method to yield the same reference void time as that in another nominally identical method substantially reduces the retention time differences in all such methods. This process, known as retention time locking (RTL), can also be viewed as a reverse method translation where, instead of the rescaling the temperature program to adopt to the changes in the void time, the latter is adjusted to adopt to a fixed temperature program. A theoretical study of RTL can be found in Blumberg, L. M. and Klee, M. S., xe2x80x9cMethod Translation and Retention Time Locking in Partition GCxe2x80x9d, Analytical Chemistry, vol. 70, number 18, Sept. 15, 1998, pp. 3828-3839. Several implementations of RTL are described in U.S. Pat. Nos. 5,827,946, 5,987,959, 6,036,747, and 6,153,438.
Benefits of Conventional Method Translation
Conventional method translation allows an increase in the speed of analysis (some times by a factor of 10 or more) with no or little additional method development while preserving the solute elution pattern and resolutions of all peak pairs. Method translation also allows a trade-off of separation for time and vice versa, or either of those for the sample capacity, etc.xe2x80x94all without affecting the solute elution pattern and requiring little or no additional method development. (In the original method development, this can be used for fast repetitive trials until the best conditions are found and then upgrading the final conditions to achieve a required separation. In testing of large numbers of samples, it can be used for fast screening with lower resolution followed by more accurate analysis of only the selected samples.) Method translation also facilitates porting of GC methods from one set of conditions (different instruments, different columns, different ambient environment, etc.) to another. Method translation may be made to appear transparent. All mutually translatable methods can be expressed via a unique normalized temperature program (a temperature program expressed in terms of dimensionless time) which can be treated as a core of all mutually translatable methods.
A transparent pneumatic optimization (optimization of a carrier gas flow rate, velocity, pressure, etc.) of a column may be performed in isothermal or temperature programmed analysis in order to achieve the best tradeoff between the shortest possible analysis time and adequate separation of a given (typically the most critical) pair of peaks without any change in a peak elution pattern. In addition to that, Retention Time Locking (RTL) is just one of several implementations of method translation.
Shortcomings of Conventional Method Translation
Unfortunately, the currently known method translation algorithms are very restrictive. The key requirement for the currently known method translation and RTL algorithms is that the ratio, tM1/tM2, of the void times measured at the same temperature in two mutually translated methods 1 and 2, respectively, be the same for all temperatures. This leads to the requirement for constant pressure mode in order for a method to be translatable.
One of the most practically important departures from the constant pressure mode is a constant flow mode of a GC analysis where the carrier gas flow rate measured at some predetermined conditions (typically, atmospheric pressure, and 0xc2x0 C. or 25xc2x0 C.) remains constant during a temperature programmed analysis. Constant flow mode has many advantages over the constant pressure mode. As a carrier gas viscosity increases with temperature, the column pressure increases with temperature in order to maintain the constant flow. Most contemporary GC instruments are equipped with the electronic pressure control (EPC) regulators that allow implementation of constant flow mode. Unfortunately, currently known method translation and RTL algorithms do not preserve the peak retention pattern (the main task of the method translation) in a constant flow mode.
Occasionally, pressure programming is used to further speed-up a temperature programmed analysis. Unfortunately, currently known method translation and RTL algorithms might not work in the presence of an arbitrary pressure program.
Furthermore, the key requirement of the temperature-independence of the ratio, tM1/tM2, imposed by currently known method translation and RTL algorithms leads to the requirement that the ratio, xcex71/xcex72, of viscosities, xcex7, of different types of the carrier gas used in two mutually translatable methods 1 and 2, respectively, be independent of temperature. This narrows the choice of the types of the carrier gas that allow the use of the currently known method translation algorithms.
Previously known method translation techniques in gas chromatography (GC) only translated between constant pressure mode methods where column pressure remains fixed during the analysis. Due to this limitation and other shortcomings of known method translation techniques, there exists a need for a method translation technique that can translate between methods, regardless of whether the methods are run in constant pressure mode.
The present invention is directed to a new GC method translation technique that eliminates the need for a constant pressure mode, the need for a temperature-independent ratio of carrier gas viscosities, and other shortcomings of the currently known method translation techniques and RTL techniques. Elimination of these restrictions comes from the elimination of the key requirement of the currently known method translation and RTL algorithms that the ratio, tM1/tM2, of void times, tM1 and tM2, measured at the same temperature in two mutually translated methods 1 and 2, respectively, be temperature-independent.
Two mutually translatable GC methods 1 and 2, subject to the new method translation technique, may have void times tM1and tM2, respectively, that change independently during the analysis.
The basis for method translation is preservation of the elution temperatures, Te, of all solutes of interest. The latter is the column temperature at retention time, tR, of the peak corresponding to the solute. Two methods are mutually translatable if, in both methods, any solute elutes at the same temperature. While the emphasis of the previously known method translation and RTL algorithms was prediction and preservation of retention times, the new technique focuses directly on preservation of the solute elution temperatures. This, in turn, leads to a more universal method translation techniques that allows an arbitrary pressure program during a temperature program.
An elution pattern for a given sequence of solutes is a sequence of respective elution temperatures of these solutes. All mutually translatable methods yield the same solute elution pattern for the same mixture. With the focus in the new method translation on the elution temperature (rather than the retention times as in the previously known techniques), the previously known RTL (retention time locking) is being replaced by elution pattern locking (EPL). The latter is more universal and easier to maintain than the former.
When two mutually translatable temperature programmed GC analyses are allowed to have mutually independent pressure programs, the only leverage for maintaining the same elution temperature for each solute in both analyses is adjusting the heating rate in the translated analysis. As a result, a translation of a linear temperature ramp (i.e. the one that has a fixed heating rate) might be a temperature vs. time curve where the heating rate gradually changes with time. Most commercially available GC system are designed to provide only a limited number (typically 3 to 6) of linear temperature ramps preceded and/or followed by temperature plateaus. In order to take a full advantage of the new method translation and EPL, a GC system should be capable of a transparent gradual variation of a column temperature (i.e., a temperature vs. time curve). This can be implemented in the same manner as the column pressure is changed during the analysis to maintain a constant flow rate.
According to one aspect of the present invention, translation may be performed to and from the methods with arbitrarily different pressure and flow programs. This includes the translation of pressure programmed methods into the flow programmed methods and vice versa, translation of a constant pressure mode into a constant flow mode (carrier gas flow remains fixed during the analysis) and vice versa, translation from the constant flow mode with one flow rate to the constant flow mode with another flow rate, etc.
According to another aspect of the present invention, translation to and from the methods may be performed with a wider choice of carrier gas types.
According to a further aspect of the present invention, all methods can be implemented as different specific executions of the same normalized temperature program. No explicit translation of one method into another is necessary. Elution pattern locking is automatic.
According to yet another aspect of the present invention, a method can be designed to generate a solute elution pattern that is unique for all mutually translatable methods. This allows elimination of the need for a change of a calibration table when method parameters (column dimensions, carrier gas type, pressure program, etc.) are changed.
According to a further aspect of the present invention, GC system resources may be better utilized. If, for example, an oven heating rate required by a temperature program exceeds a maximum available in a given instrument, a heating (linear or ballistic) rate that is lower than the required rate can be allowed. In order to preserve the elution pattern, the pressure program can automatically compensate for the departure of the temperature program from its nominal settings. This allows the use of temperature programs that exploit the actually available maximum heating rate instead of the guaranteed maximum heating rate which is, typically, substantially lower than the actually available one.
According to yet another aspect of the present invention, a transparent pneumatic optimization (optimization of a carrier gas flow rate, velocity, pressure, etc.) of a column in isothermal or temperature programmed analysis may be performed in order to achieve the best tradeoff between the shortest possible analysis time and adequate separation of several (typically the most critical) pairs of peaks.
According to a further aspect of the present invention, Elution Pattern Locking (EPL) for an arbitrary pressure/flow regulation includes constant pressure and constant flow modes.